Abstract

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.

(A) Stereoview of TssJ in ribbon representation and rainbow coloring, from blue (N-term) to red (C-term); the sequence is represented above. Figure made with Pymol . (B) Topology cartoon of TssJ (same coloring as in (A)). (C) Structural comparison of TssJ and its nearest homologue, transthyretin (1sn5), after superimposition. The topology is identical for both proteins and their β-sandwiches superimpose within 3.2 Å. Note the presence of an extra helical domain in TssJ, and an extra helix (top) in transthyretin.

(A) Solubilized extracts of E. coli K12 W3110 strain producing (+) or not (-) HA-tagged TssJ and FLAG-tagged TssM-ekto or -Nt or –Ct derivatives were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total solubilized material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS PAGE, and immunodetected with anti-HA (TssJ; lower panel) and anti-FLAG (TssM-ekto and sub-domains; upper panel) monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (B) Gel filtration showing the direct interaction of TssM-ekto with TssJ. The SDS-PAGE analysis of the fractions is shown on the left panel. The chromatogram of the gel filtration is shown on the right panel. (C) MALS/QELS/UV/RI analysis of the TssM-ekto/TssJ complex.

Measure of the interaction between TssM-ekto and TssJ by Surface Plasmon Resonance.

(A) Sensorgram and saturation curve of the titration of Trx-TssJ by Trx-TssM-ekto. The CM5 chip (BIAcore) was coated with TssJ N-terminal thioredoxine fusion with 600 response units (RU) and the Trx-TssM-ekto was injected in the microfluidic channel. (B) Sensorgram and saturation curve of the titration of Trx-TssM-ekto by TssJ. The CM5 chip was coated with TssM-ekto N-terminal thioredoxine fusion with 3000 response units, and TssJ was injected in the microfluidic channel. The KD values were obtained using the fitting tool of the BIAevaluation software (BIAcore).

The outer (OM) and inner membranes (IM) are represented in light green. The T4 phage-like central puncturing device includes Hcp (green disks) and VgrG (purple). The “tail sheath” TssBC (VipAB) proteins are shown in blue, around the central Hcp/VgrG pilum. The TssBC proteins constituting a sheath encompassing the Hcp tube has not been evidenced but is speculated based on the similarities between the T6SS TssBC subunits and the bacteriophage T4 sheath , . The three-transmembrane inner membrane TssM protein (yellow) interacts with the TssL IM protein (blue) . TssL interacts with TagL (green), an IM protein that anchors the T6SS to the cell wall . TssM C-terminal domain interacts with a loop of the outer membrane lipoprotein TssJ [this study]. In this model, the TssL-TagL-TssM-TssJ complex forms a trans-envelope spanning channel.